The lower barriers for h-BN are mainly due to boron atoms. The trends of kinetic barriers are B < BN < N-doped graphenes. The permeation selectivity of graphene models increases with doping. Our studies suggest that boron-doped graphene models show an energy barrier of 0.04 eV for the permeation of proton, much lower than that of the model graphene and h-BN sheet, while nitrogen-doped graphenes have a very high energy barrier up to 7.44 eV for permeation. Therefore, boron-doped graphene models are suitable candidates for proton permeation. Moreover, the presence of carbon atoms in the periphery of BN sheets has significant negative effects on the permeation of proton isotopes, an unexplored dimension of the remote neighboring effect in 2-D materials. This study illustrates the need for permeation study through other hetero-2D surfaces, where interesting hidden chemistry is still unexplored.Two-dimensional (2D) monolayer nanomaterials are the thinnest possible membranes with interesting selective permeation characteristics. Among two-dimensional materials, graphenes and hexagonal boron nitride (h-BN) are the most promising membrane materials, which can even allow the separation of proton isotopes. The current work aims at understanding the higher reported permeability of h-BN by sequential doping of B and N atoms in graphene nanoflakes. The kinetic barriers were calculated with two different models of graphenes; coronene and dodecabenzocoronene  Two-dimensional (2D) monolayer nanomaterials are the thinnest possible membranes with interesting selective permeation characteristics. Membranes are useful in separation processes such as water purification, waste water treatments and desalination of ocean water.2 Moreover, they can also be used for the purification of different gases.3 However, the membranes also possess a number of medical applications such as membrane oxygenation, hemodialysis, artificial kidney, artificial pancreas, artificial liver and bioreactors.4 Furthermore, biological membranes present in the living cells are responsible for the selective transport of ions and other useful polar molecules across the lipid bilayer.5 The membranes may be natural or synthetic.Membranes are selectively permeable boundaries that allow the passage of certain particles through it while restricting others due to a specific driving force.6 Natural membranes are very much important for the normal functioning of all the cells. They perform functions quite rapidly with less energy consumption, commonly via active transport.7 These membranes regulate the rate of important information transfer among cells either by identifying received pulse molecules from different cells, or through forwarding electrical and chemical signals to the cells.8 On the other side, synthetic membranes are not as complicated in their anatomy and functions as natural membranes. These membranes possess only nonresistant transport characteristics and these are mostly less selective and energy-saving. Examples of synthetic membranes include inorganic,9 polymeric,10 mixed matrix,11 and carbon molecular sieve membranes.12 In general, they contain effectively enhanced chemical and mechanical stability, principally at high temperatures. Their selectivity is analyzed through a sieve-like structure according to their capacity or through a homologous structure according to the solubility and diffusivity of solutes.13 Synthetic membranes play an important role in reverse osmosis, filtration processes, pervaporation, emulsion liquid membranes, solvent extraction through membranes, various membrane reactors, gas permeation and supported liquid membranes.14 Among the synthetic membranes, graphene membranes are found to be very attractive for scientists because of their extraordinary properties.Natural or biological membranes are responsible for complicated and selective transport within the living beings. These membranes are highly selective, thereby controlling the incoming and outgoing transport of all particles, maintaining the conformation of the cellular fluid.15 Thus, the synthetic production of nano pores by various methods such as electron beam treatment or production of feasible initiators for the self-building of graphene structures is examined.16 Some computational studies were performed on the formation of pores in graphene sheets for important applications such as separation of H2 from CH4, separation of helium from other noble gases and CH4, specific transport of ions, DNA characterization and H2O filtration.17 In all separation procedures based on the graphene and other porous membranes, sieving is a feasible process that permits the passage of particles smaller than graphene pores, but stops the transport of larger ones.18 For example, quantum mechanical studies explained that graphenes with a pore size of about 3.3 Å show greater specificity (high perforation rate) to hydrogen than to other gases.19 This specific sieving property can be generally postulated as the greater surface adsorption of bigger particles by graphene sheets, known as the preferential surface adsorption. This feature can boost the passage of particles via mono-layered graphene membranes.20 In addition to graphene monolayers, there are certain other 2D analogues of graphenes that have been reported, which include hexagonal boron nitride (h-BN),21 borophene,22,23 stanene,24 silicene,25 black phosphorus,26 germanene,27 and transition-metal dichalcogenides .28 Recently, 2D crystals of metal organic frameworks (MOFs),29 covalent organic frameworks (COFs),30 and MXenes31 have also been reported in the literature, and are found to exhibit remarkable properties similar to graphenes. Moreover, the one atom thickness of graphene monolayers and their analogues makes them the thinnest membranes with an extraordinary specific staining feature for the separation of gases and dissolved ions.32,33However, an ideal layer of graphene (non-defected) is not permeable for gas atoms (even for He atoms), which are repulsed due to the electron density of the graphene's aromatic rings.34 Recently, it has been reported that the monolayer of graphenes and h-BN exhibit subatomic selectivity, i.e., easier permeation of protium ions (H+) compared to heavier deuterium ions (D+).35 Geim et al. discovered that these 2D single-layered membranes are able to separate proton isotopes such as protium and deuterium. They demonstrated that the dissimilarity in the transportation velocity is due to the difference in zero-point energies, which provides a comparatively high selectivity ratio for these two isotopes. The sieving energy barriers (Eb) calculated experimentally for graphenes and h-BN are 0.8 eV and 0.3 eV, respectively.36 Furthermore, the separation factor was estimated to be about 10 for H+/D+. This factor is much larger than that of the previously reported factor of <2.5.37 Moreover, it has been shown that the permeation of proton through the 2D nanomaterials is thermally activated. This isotopic separation is based on velocity when transported by a single layer of graphene membranes. In addition, it has been reported in the literature that the doped graphene permits rapid permeation of protium and deuterium ions as compared to pure and normal graphenes.34 Separation of isotopes is of great importance for various analytical and tracing technologies, and thus, makes 2D graphenes and their analogues very promising for separating proton isotopes.These 2D single-layer nano-sized materials are utilized as the thinnest membranes with selective staining characteristics for H-isotopes.et al. theoretically studied the permeability of proton isotopes through graphenes and h-BN while using small coronene and its analogue models. Their results were consistent with the experimental studies that these nanomaterials are selective for protium than deuterium and tritium. Moreover, the permeability of h-BN is higher than that of graphenes. This study provides rationalization for the high permeability of h-BN than graphenes, but it does not unveil the specific effects of boron and nitrogen atoms. We became interested in studying the effect of boron and nitrogen atom doping in graphenes on the permeability for proton isotopes. In this regard, multiple boron, nitrogen and BN (co-doped) atoms are doped on the coronene model.Zhang 2+), deuterium (D+) and tritium (T+)) through two-dimensional graphene membranes.36 All calculations were performed using the Gaussian 09 package.38 All results were visualized using a graphical interface such as GaussView 5.0.39 Frequency analysis was performed by the ωB97XD method of DFT with the 6-31++G basis set. ω B97XD is a range separated hybrid functional.40,41 It was one of the first DFT methods that had a significant improvement over Hartree–Fock. H+, D+ and T+ have the same electronic structures. Therefore, energies of H+, D+ and T+ are the same. Because of this reason, these isotopes cannot be differentiated based on electronic energies. For their differentiation, zero-point correction and Gibbs free energies are calculated. In this work, two models were taken for graphenes, i.e. coronene and dodecabenzocoronene (DBC) and doping with boron and nitrogen was studied for proton isotope permeation. Moreover, N, B, BN, doped coronene and dodecabenzocoronene were also used to study the effect on the proton permeation rate. Furthermore, zero-point correction difference was also studied for selectivity ratios for H+, D+ and T+ isotopes to understand the permeability behavior in various cases.Density functional theory (DFT)-based methods are used to study the permeation of proton isotopes . The lowest activation barrier of H+ indicates its high permeation rate through coronene as compared to D+ and T+. This may be attributed to the difference in ZPE between the isotopes.35 The lighter H+ possesses greater ZPE compared to the heavier isotopes and, hence, faces a lower barrier.First, a pure coronene model was taken to evaluate the permeability of proton isotopes. It is selected since it resembles the graphene fragment, and this model has been used previously in the theoretical studies. The hexagon present in the center has a pore environment very similar to the hexagonal ring in the graphene monolayer. In this study, the proton isotopes were individually placed at a vertical distance of ∼2 Å normal to the central pore and were allowed to approach the cavity. It was observed that, as the proton approaches towards the center, the total energy of the system decreases gradually up to ∼1 Å. On further translation, the total energy begins to rise, and the maximum energy is obtained when the proton is placed exactly in the center of coronene . All theV for D+ . The hig+ as a reference, while zero-point corrected values of other two isotopes were subtracted from the reference value, i.e. EZPE(H+)–EZPE(D+) and EZPE(H+)–EZPE(T+), respectively . The selectivity ratios K+H/K+D and K+H/K+T for proton isotopes are 1.27 and 1.40, respectively. The results indicate that the heavy isotopes, deuterium (D+) and tritium (T+) permeate slower than lighter protium (H+) isotopes through the coronene model.In order to verify this concept, zero-point energies for all proton isotopes were computed using the same level of theory. Zero-point correction differences were calculated by taking Hectively . The difectively . Based o3.22 hybridized boron and nitrogen atoms. It has been reported experimentally by Geim and his co-workers that a monolayer of these nanomaterials favorably allows the transport of proton with a relatively lower thermal activation barrier of ∼0.3 eV.35 Similar to the coronene model, the h-BN fragment was taken to calculate the permeation barriers for proton isotopes. It was observed that the total energy of the system continuously decreases as the proton isotopes are approaching towards the central pore. However, from a distance of ∼1 Å, the energy begins to rise (a phenomenon similar to coronene permeation). The calculated energy barriers for H+, D+ and T+ are 0.65 eV, 0.66 eV and 0.67 eV, respectively  sheet is another two-dimensional nanomaterial, which contains a network of spectively . These bgraphene .+ and T+ were 0.019 and 0.041 eV mol−1 with respect to that of H+, respectively (+H/K+D and K+H/K+T) values for D+ and T+ with respect to H+ are 2.18 and 4.84, respectively. These ratios are lower than those calculated for coronene. The results have indicated that the proton isotopes pass through the h-BN sheet with a greater velocity than that in case of pure graphenes, with a slightly lower selectivity.Similar to the pure coronene, zero-point correction differences were also calculated for the h-BN sheet. The calculated differences for Dectively . Moreove3.342 The larger radius of the boron dopant as compared to that of carbon is responsible for the increase in C–B bond length.43 Thus, a disorder is produced in the hexagonal lattice by placing dopants at central rings.44In order to enhance the permeation properties of graphene sheets, these materials were modified by doping. Therefore, the coronene sheet was further modified by substitutional doping. An ideal dopant should not affect the planarity of coronene. The boron element is a more important dopant, which can induce new and complementary properties useful for alternative devices and technologies such as sensors and photovoltaics.+ was reduced to 0.77 eV compared to what it was in pure coronene (1.08 eV)  . Similarcoronene .+ is 0.63 eV. The calculated energy barriers for D+ and T+ are 0.64 and 0.65 eV, respectively  are 0.46, 0.47 and 0.48 eV for H+, D+ and T+, respectively. When four boron atoms are incorporated in the hexagon ring, the barriers are further reduced to 0.18, 0.19 and 0.20 eV for H+, D+ and T+, respectively. The lowest barriers for proton permeation are observed when all the six members of the ring are replaced with boron atoms. The calculated barriers for hexa-boron-doped coronene (6B-coronene) are 0.04, 0.05 and 0.06 eV for H+, D+ and T+, respectively  and 1.24 (K+H/K+T) in 4B-coronene. The lowest selectivity ratios were calculated in 6B-coronene, which are 1.06 and 1.11 for K+H/K+D and K+H/K+T, respectively. This gradual decrease in the selectivity of doped coronene with the increase in the number of boron atoms is due to the increase in the pore size. As the number of boron atoms increase in the hexagonal ring, they offer more distortion to the ring, and hence, the size of the pore increases, which, in turn, increases the permeability. At the same time, the selectivity of the ring decreases because it will not resist that much to the heavier isotope compared to the pure coronene.The selectivity ratios for proton isotopes through doped coronene models were also calculated from zero-point correction differences. In case of B-coronene, it was observed that selectivity ratios Kectively . These s3.445 By incorporating nitrogen atoms, three different types of configurations were obtained such as pyrrolic N, pyridinic N, and graphitic N.45 Nitrogen-doped graphenes have shown significant applications in the field of biosensors and bioelectronics.46 Moreover, nitrogen doping in the hexagon of graphene sheets acts as a strong scattering center.47The incorporation of nitrogen atoms plays an important role especially in carbon-based materials.+, D+ and T+, respectively  , when prectively . These b+ and T+ is 4.48 eV while for D+, it is a bit lower, i.e. 4.47 eV, respectively. On further increasing the number of nitrogen atoms in the ring, an odd-even pattern of proton permeation through coronene models is observed (The energy barriers are slightly reduced (compared to mono-nitrogen-doped systems) when two of the nitrogen atoms are incorporated into the hexagonal ring of coronene. The calculated barrier for Hobserved . For exa+H/K+D and K+H/K+T are calculated in case of 5N-coronene . This is due to the obvious greatest permeation barriers for proton isotopes in 5N-coronene. As the size of the pore decreases, the permeation barriers increase and hence the pore gets more selective for the transport of lighter ions. The permeability and selectivity ratios are inversely related to one another. Any factor which increases the permeability leads to a decrease in selectivity and vice versa.Similar to permeation barriers, an even-odd pattern for selectivity ratios of nitrogen-doped coronene was also observed . The hig3.549 It has been reported that co doping of BN atoms can produce a variable band gap, which depends on the placement of boron and nitrogen atoms on the sheet.50 Such nanomaterials are very useful in opto-electronics applications.BN co-doped graphenes have been studied to investigate the structural and electronic properties of doped 2D materials.+ permeation, the calculated energy barrier is 1.15 eV. Whereas energy barriers for D+ and T+ are 1.16 and 1.17 eV, respectively. These energy barriers are comparatively lower than those of nitrogen-doped coronene models but higher than those of boron-doped and pure coronene models. These results indicate that the rate of isotope permeation is higher in BN-doped coronene than that of nitrogen-doped coronene but lower than boron-doped and pure coronene models  in 2(BN)-coronene sheet remained, while for T+, it is 1.21 eV, respectively. Similarly, when 3(BN) atoms are introduced into the ring, the barriers further reduced compared to 2(BN)-coronene. The potential barriers of 1.16 eV, 1.17 eV and 1.18 eV are observed for H+, D+ and T+, respectively. This shows that increasing number of nitrogen atoms in the ring are offering attractive interactions to the passing protons due to which the barriers are rising.The permeation barriers for proton isotopes are increased when 2(BN) atoms are incorporated in the central hexagon. The H+H/K+D and K+H/K+T were calculated as 1.71 and 2.10, respectively. In case of 2(BN)-coronene, these ratios are increased to 2.08 and 2.39 for K+H/K+D and K+HK+T respectively. However, the selectivity ratios are lower in the case of 3(BN)-coronene where the calculated K+H/K+D and K+HK+T are 1.38 and 1.62, respectively.Relative selectivity ratios calculated from zero-point correction differences have shown an irregular pattern. For BN-coronene, K3.651 Dodecabenzocoronene can easily accommodate the coronene analogue of BN in its geometry  is a stable aromatic compound and it is the extension of coronene molecule. It consists of nineteen benzene rings arranged as a hexagonal structure.etry see . In this+ and deuterium D+ isotopes was studied. The energy barrier for the permeation of H+ and D+ was 0.70 eV and 0.71 eV, respectively. These barriers are comparatively higher than those calculated when two boron atoms were doped on a coronene sheet. Similarly, when boron and nitrogen atoms are doped simultaneously on the DBC, the calculated barriers for H+ and D+ are again higher than those of BN-coronene  dopants, the computed energy barrier is 1.24 eV for the H+ isotope, while in the case of D+, this energy barrier is 1.27 eV. The energy barriers observed for H+ and D+ in the case of nBN-doped DBC models are lower than those of pure h-BN sheets (1.48 eV) as reported by Zhang et al.34 In pure h-BN, van der Waals radius is larger than that of the nBN-doped DBC model. The calculated radii are 1.44 and 1.41 Å for h-BN and nBN dodecabenzocoronene models, respectively. On the other side, the permeation rate is comparable in both cases (nBN-doped DBC models). Therefore, it can be safely concluded that the presence of carbon atoms on the periphery of the BN model provide additional effect for the permeation of proton isotopes. This study illustrates the need for permeation study through other hetero-2D surfaces, where interesting hidden chemistry is still unexplored.Finally, DBC is co-doped with ‘3.7+ and bare and doped coronene sheets. We performed electronic properties for H+ only, because electronic properties of D+ and T+ are identical to those of H+ doped systems (due to the same number of electrons). The charge on H+ in each case obtained through the NBO analysis is reported in Table S1.+ in case of pure h-BN, and nB-coronene models, but this charge is less than +1 , which indicate that the electronic cloud is donated by h-BN sheet and nB-coronene models. In the case of B-coronene and 2B-coronene models, charges on H+ are 0.50 e− and 0.56 e− respectively. However, a decrease in the positive charge intensity is noticed as the number of B-atoms increases in the coronene model , 3N-coronene (−0.81 e−) and 6N-coronene (0.60 e−). In the rest of the boron-nitrogen-doped coronene models, transfer of the charges is noticed from doped coronene to H+. In all designed systems, N-doped coronene models shift the highest amount of charge towards H+ compared to other doped coronene models.NBO charges were studied to find the direction of charge transfer between H+, there is irregular change in HOMO–LUMO gaps with the increase in N-atoms. However, a significant decrease in HOMO–LUMO gaps is observed in the case of 2N-coronene (3.10 eV) and 3N-coroene (3.23 eV) compared to pure coronene sheets (6.07 eV). The increase in the conductivity of H+@BN-coronene and H+@BN-DBC models is attributed to more increase and decrease in energies of their HOMOs and LUMOs, respectively.The HOMO–LUMO energy gap of 6.07 eV and 7.06 eV is observed in the case of pure coronene and h-BN sheets. The HOMO–LUMO gaps for all the doped coronene models are given in Table S1.4+, D+ and T+ has been studied through boron- and nitrogen-doped graphene membranes. The permeation barriers of undoped graphenes (coronene model) are higher than those of boron-doped analogues. This is because the van der Waals radii are greater in undoped coronene models, which decreases the pore size for proton permeation. However, boron-doped coronene models have smaller van der Waals radii, which increase the pore size for proton permeation. Moreover, by increasing the number of boron dopants from 1 to 6 atoms, a decrease in permeation barrier was observed. For boron doping, the highest energy barrier (∼0.77 eV) is observed in mono-boron-doped coronene models, whereas the lowest energy barrier (∼0.04 eV) was observed in hexa-boron-doped coronene models. Therefore, the permeation of proton is faster in hexa-boron-doped coronene models. In p-type materials, the formation of holes is also a reason for the fast permeation of proton isotopes. An odd-even pattern of ZPE was obtained by nitrogen doping for proton permeation through coronene models due to the unfavorable radical effect of doped nitrogen atoms. The observed order of permeation rate is 1 < 2 < 4 < 3 < 6 < 5 in nitrogen-doped models. BN co-doped models also show an odd-even pattern in ZPE. The order of permeation rate is 1 < 3 < 2 in BN-doped models because of unfavorable interactions of nitrogen atoms with proton isotopes. Furthermore, we studied the isotope permeation through higher analogue DBC. Most of the trends are similar to those of the coronene model; however, the selectivity reversal is also observed in nBN- and 2(BN)-doped DBC models due to unfavorable interactions of nitrogen atoms. The permeation rate of proton isotopes is lower in DBC models than that of coronene models due to greater interactions in DBC models. Our studies suggest that boron-doped graphene models show a very low energy barrier of ∼0.04 eV for the permeation of proton compared to the pure graphene model and h-BN sheets, while nitrogen-doped graphenes have a very high energy barrier up to 7.44 eV for permeation. Therefore, boron-doped graphene models are suitable candidates for proton permeation. Moreover, the presence of carbon atoms in the periphery of BN sheets has significant negative effects on the permeation of proton isotopes, an unexplored dimension of remote neighboring effect in 2-D materials. This study illustrates the need for permeation study through other hetero-2D surfaces, where interesting hidden chemistry is still unexplored.The permeation of proton isotopes HThere are no conflicts to declare.RA-012-D1RA09398C-s001


Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in the world with a high mortality rate. The mechanism about ESCC development and whether miRNAs play a critical role remains unclear and needs carefully elucidated.High-throughput miRNA sequencing was used to identify the different expression miRNAs between the ESCC tissues and paired adjacent normal tissues. Next, both CCK-8, Transwell and apotosis assay were used to evaluate the role of miRNA in ESCCcells. In addition, we used bioinformatic tools to predict the potential target of the miRNAs and verified by Western Blot. The function of miRNA-target network was further identified in xenograft mice model.in vitro and in vivo experiments.In ESCC, we identified two miRNAs, miR-17-5p and miR-4443, were significantly upregulated in ESCC tissues than adjacent normal tissues. TIMP2 was proved to be the direct target of both two miRNAs. The miR-17-5p/4443- TIMP2 axis was shown to promote the tumor progression This study highlights two oncomiRs, miR-17-5p and miR-4443, and its potential role in ESCC progression by regulating TIMP2 expression, suggesting miR-17-5p and miR-4443 may serve as a novel molecular target for ESCC treatment. Esophageal cancer (EC), one of the most frequently diagnosed cancers in the world, has the highest incidence rate in Eastern Asia . It has MicroRNAs (miRNAs), which consisting of 18-22 nucleotide base pairs, has been verified to play important roles in regulating gene expression . The abnIn this study, we identified the overexpression of miR-17-5p and miR-4443 in EC tissues compared to their paired adjacent tissues. Overexpression of miR-17-5p and miR-4443 promote EC cells’ proliferation and migration as well as reduces the expression of TIMP2, while down-regulation of miR-17-5p and miR-4443 got the opposite effect. We testified the important roles of miR-17-5p and miR-4443 in the development of EC and these results may provide new strategies for ESCC treatment.20 ESCC tissue samples and their paired adjacent paratumor normal tissues were collected from Jiangsu Cancer Hospital . All patients signed the informed consents and the Ethics Committee of the Jiangsu Cancer Hospital approves the whole study. The protocol used in this study was based on approved guidelines by Ethics Committee of the Jiangsu Cancer Hospital. All patients were diagnosed ESCC by histopathology examination, and none of them had diagnosed other malignant tumor or received neoadjuvant chemotherapy or radiotherapy. All samples were immediately cut into small pieces after surgical resection and keep in liquid nitrogen until use.The human esophageal cancer cell line, TE-10, and ECA-109 were purchased from Cell Bank of Chinese Academy of Science, Shanghai, China. All cells were cultured in RPMI-1640  containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100ug/mL streptomycin. The humidified incubator was set at 37°C containing 5% CO2.Total RNA extracted from 3 ESCC tissues and its paired adjacent normal tissues were used for high-throughput miRNA sequencing. The detailed procedure was the same as previously described .-△△CT method was used to compare the relative quantification of each miRNA between every group.Total RNA was extracted from cells or tissues by TRIzol reagent (Invitrogen) and quantified by NanoDrop spectrophotometer. TaqMan miRNA probes  were used to quantify the miRNAs. All procedures were performed as previously described . And allAll of the miRNA mimics, inhibitors and scrambled negative control used in this research were designed and synthesized by GenePharma . The sequence of mature miR-17-5p and miR-4443 are 5’-CAAAGUGCUUACAGUGCAGGUAG-3’ and 5’-UUGGAGGCGUGGGUUUU-3’, respectively. Lipofectamine 2000  was used as the cell transfection reagent and performed according to the manufacturer’s instructions.3 cells per well. A Cell Counting Kit-8 assay (CCK-8) was performed at 0, 24h, 48h, and 72h respectively. The absorbance of the 450nm laser was measured after 2-hour incubation of cells and CCK-8. Each group had at least 5 repeats and all experiments were performed in triplicate.TE-10 and ECA-109 cells were seeded in the 6-well plate. 6h after transfection, the cells were reseeded to a 96-well plate at a density of 5×105 cells suspended with serum-free RPMI-1640 were added into the upper chamber and 500μl RPMI-1640 with 10% FBS was added to the lower chamber. After 16h incubation, the cells in the upper chamber was wiped with a cotton swab and the cells migrated to the lower surface were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. The stained cells were then quantified by a spectrophotometer at 3 random areas. The apoptosis of cancer cells was tested by Annexin V-FITC/PI staining kit . Besides, the total apoptotic cells were counted as the sum of early apoptotic (PI− AV+) and late apoptotic (PI+ AV+) cells.Transwell assay was used to test the cells migration ability. In brief, the transwell chamber with 8μm pore polycarbonate membranes was put into a 24-well plate. A total number of 1×10The 3′-UTR of human TIMP2 containing putative binding sites was cloned into the p-MIR-REPORT plasmid (Ambion), and efficient insertion was confirmed by sequencing. To test the binding specificity, the sequences in human TIMP2 3′-UTR that interact with miRNA seed sequence were mutated. 293T cells were co-transfected with β-galactosidase  expression plasmid (Ambion), a firefly luciferase reporter plasmid, and miRNA mimics or negative control. The β-gal plasmid was used as a transfection control. Luciferase activity was measured 24 h after transfection using a luciferase assay kit .The expression of TIMP2, as well as internal control GAPDH in cells and tissues, was assessed by western blot analysis. Homogenate tissues and cultured cells were lysed in RIPA buffer containing protease inhibitor cocktail. We used the 10% SDS-PAGE gels to separate the protein lysates, which was then electrically transferred to a polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked by 5% skimmed milk for at least 1h at room temperature and followed on incubating with primary antibody . After incubating with their specific second antibody at room temperature for 1h, the membranes were then visualized by ECL  detection assay.6 cells per 0.2ml PBS. 28 days later, the mice were sacrificed and removed the xenografted tumors. The tumors were then measured the volumes and weights and then extracted protein for the TIMP2 expression detection.All animals used in this study were approved by the ethics committee of Jiangsu Cancer Hospital and complied with NIH Guidelines. TE-10 cells were treated with miR-17-5p/miR-4443 overexpressing lentivirus or control lentivirus and were then injected subcutaneously into the inguinal folds of the nude mice at the concentration of 10All western blot images are representative of at least three independent experiments. Quantitative RT-PCR, luciferase reporter assay, cell proliferation, migration assay and cell apoptosis assay were performed in triplicate, and each experiment was repeated several times. Statistical analysis was calculated by SPSS 16.0. Presented data was carried out by at least 3 separate experiments and showed as mean ± SD. P<0.05 was considered statistically significant in this study by using the student’s t-test. *P<0.05, **P<0.01, ***P<0.001.To explore the significantly expressed miRNAs in ESCC, we first use the high-throughput miRNA sequencing to identify the expression profiles of all miRNAs in the ESCC tissues and paired adjacent normal tissues. As shown in To further explore the specific role of miR-17-5p and miR-4443 in the ESCC, we transfected the TE-10 and ECA-109 cells with miRNA mimics, inhibitors, and negative control then checked their effects on tumor behavior. As shown in http://www.targetscan.org/vert_72/ and miRDB http://mirdb.org/). Because of miR-17-5p and miR-4443 having a similar effect on ESCC, we hypothesize if they could target the same protein. As shown in To identify the direct target genes of miR-17-5p and miR-4443 in ESCC, we used two bioinformatics tools , was considered to be a potential target due to the known functions in cell proliferation and migration. Other target genes may also contribute to the effect of miR-17-5p and miR-4443 in ESCC cells. Among all results, TIMP2 was experimentally validated to be down-regulated by both of the miRNAs. Clinical ESCC tissues also showed lower expression of TIMP2 than adjacent non-carcinoma tissues. These results suggested that TIMP2 may serve as a tumor suppressor and be down-regulated during tumorigenesis, as has been shown by other researches  is a member of the tissue inhibitor of metallopeptidases (TIMPs). The metastasis of cancer cells should invade into the extracellular matrix (ECM) firstly, and matrix metalloproteinases (MMPs) are essential and play core effect to degrade the ECM, paving a road for tumor cells to migrate into cycle system for distant metastasis . On the Taken together, our research demonstrated that miR-17-5p and miR-4443 are significantly upregulated in ESCC tissues, and serve as a tumor promoter by directly targeting TIMP2. Ectopic expression of miR-17-5p and miR-4443 may be one of the reasons for the up-regulation of MMP-2 in ESCC tissues. And the unbalanced state between TIMP2 and MMP-2 promote ESCC development and distant metastasis. Our research develops a new approach for understanding ESCC development and miR-17-5p and miR-4443 may serve as a potential target for ESCC therapy in future.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the Jiangsu Cancer Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ethics Committee of the Jiangsu Cancer Hospital.XC and LX planned the study. XW, JYH, and YL carried out the experiments. JWH and ML performed the data analysis and helped to draft the manuscript. XW wrote the manuscript. All authors contributed to the article and approved the submitted version.The reviewers YW and YX declared a shared affiliation with author XC, to the handling editor at time of review.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants of public health concern. Multiple biological mechanisms have been hypothesized to contribute to PAHs-associated adverse health effects. Little is known about the impact of PAHs on endocrine stress and inflammation in adolescence. We examined 393 Flemish adolescents (14–15 years) cross-sectionally, measured urinary concentrations of hydroxylated naphthalene, fluorene, phenanthrene and pyrene metabolites, and calculated the sum of all measured metabolites. We determined hair cortisol concentration (HCC) as endocrine stress biomarker, leucocyte counts and neutrophil–lymphocyte ratio (NLR) in peripheral blood as inflammatory biomarkers, and urinary 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) concentration as oxidative stress biomarker. Exposure–response associations were analyzed by multiple regression, adjusted for a priori selected covariates. A doubling of 1-hydroxypyrene concentration was associated with a factor of 1.13  increase in HCC and a factor of 1.07  increase in 8-oxodG. Doublings of 2- and 3-hydroxyphenanthrene concentrations were associated with a factor of 1.08  and 1.06  increase in 8-oxodG, respectively. Doubling of 2-hydroxyphenanthrene and of the sum of 2- and 3-hydroxyfluorene was associated with, respectively, a factor of 1.08  and 1.06  increase in NLR. Our results indicate the glucocorticoid pathway as a potential target for PAH exposure in adolescents and suggest oxidative stress, endocrine stress, and inflammation in adolescence as underlying mechanisms and early markers for PAH-related adverse health effects. Polycyclic aromatic hydrocarbons (PAHs) are a large class of widespread environmental pollutants generated by incomplete combustion of organic materials, such as coal, oil, wood, and gas . The genEnvironmental exposure to PAHs has primarily been linked to cancer, in particular skin and lung, bladder, and gastrointestinal cancers ,8. FurthThe biological mechanisms that contribute to PAHs-associated chronic health effects are still under investigation but are likely to be multifactorial. Toxicity of PAHs may in part be caused by reactive PAH intermediates that are formed during metabolism and that generate oxidative stress, which in turn can result in systemic inflammation and chronic disease pathogenesis ,13. In eImportantly, the health effects of environmental exposures also depend on the time in the life span when exposure occurs. Adolescence is a developmental period with increased vulnerability to environmental exposures . The ongThe aim of this study, conducted in the framework of the fourth Flemish Environment and Health Study (2016–2020), was to investigate associations of urinary OH-PAHs with early pathophysiological mechanisms that may underlie PAHs-associated health effects in a general population of Flemish adolescents, in particular chronic endocrine stress, inflammation, and oxidative stress.We hypothesized that internal exposure to PAHs is associated with an increase in chronic endocrine stress in adolescents, together with simultaneous increases in oxidative stress and inflammation. Traditional assessment of HPA axis function through measurement of cortisol levels in saliva, blood, or urine reflects cortisol concentrations over minutes to 24 h prior to sampling. Longer-term cortisol concentration, as an indicator of chronic endocrine stress, is difficult to evaluate using these matrices due to circadian variations in cortisol secretion and the need for multiple sampling . The haiTotal and differential leucocyte counts in peripheral blood are widely-used hematological biomarkers of inflammation  and haveWe simultaneously evaluated associations of urinary concentrations of PAHs metabolites in Flemish adolescents (14–15 years) with (1) the cortisol concentration in a 3 cm scalp-near hair segment, reflecting circulating cortisol levels in a 3-month period prior to sampling; (2) total and differential leucocyte count  and NLR in peripheral blood; (3) urinary 8-oxodG concentration. Given the widespread environmental exposure to PAHs and the vulnerability of adolescents to PAH-associated adverse health effects, a better insight into early pathophysiological mechanisms that may be related to PAH exposure in adolescents is of global public health importance.This study was embedded in the fourth Flemish Environment and Health study (FLEHS-4), a human biomonitoring study including 428 adolescents from the general population of Flanders. The FLEHS-4 study protocol was approved in June 2017 by the Antwerp University Hospital Ethics committee (Belgian registration number B300201732753). A stratified clustered multi-stage sampling strategy was applied to enroll equal numbers of participants across both sexes and to represent all educational levels and all Flemish provinces proportionally to the general Flemish population. The inclusion criteria of the FLEHS-4 study were informed consent signed by the adolescent and a parent, lived in Flanders for at least 5 years, and adolescent and parents mastered enough Dutch to fill out extensive questionnaires. Exclusion criteria were data of more than one questionnaire missing, blood and urine sample missing, being held back in school for more than one year, or attending a boarding school. Adolescents that attend boarding school spend a large part of their time away from home and are therefore less representative of environmental exposures in the residential surroundings. One participant was excluded from FLEHS-4 because of pregnancy. FLEHS-4 participants were excluded from the study if no data were available of urinary PAH-metabolites, urinary specific gravity, leucocyte count, or HCC. One participant was excluded from the study because of following a growth hormone therapy and a second because of a clinically elevated leucocyte count . The final study population consisted of 393 adolescents. The study was conducted according to the criteria set by the declaration of Helsinki [Between September 2017 and June 2018, participants were examined in their schools by trained nurses. Weight and height were measured using calibrated equipment. From this information, the body mass index (BMI) was calculated. Boys and girls were classified as underweight, normal weight, overweight, or obese according to the sex- and age-specific 2004 Belgian growth curves . BeforehData on the local ambient temperature at the time of sampling were provided by the Belgian Royal Meteorological Institute (KMI) that measures temperature in 15 weather stations across Flanders. We used data from the weather station closest to the adolescents’ homes. Based on the participants’ geocoded home addresses, we assessed the Area Deprivation Index (ADI) at the sub-municipality level as an indicator of neighborhood SES, as previously described . Spot urine samples were collected during field work in clean polyethylene (PE) containers and were processed immediately. To determine the specific gravity of the urine samples, 2 mL was aliquoted in a polypropylene tube, kept at 4 °C, and analyzed within 48 h after collection by refractometry at the accredited medical laboratory A.M.L. . For analysis of urinary levels of hydroxylated metabolites (OH-PAHs), 2.5 mL of urine was aliquoted in polypropylene corning tubes, subsequently frozen and stored at −20 °C until analysis. We measured urinary levels of hydroxylated metabolites of four PAHs, from here on referred to as OH-PAHs. The analyzed OH-PAHs, with their limits of quantification (LOQ), were the pyrene metabolite 1-hydroxypyrene , the naphthalene metabolite 2-hydroxynaphthalene , the sum of fluorene metabolites 2- and 3-hydroxyfluorene  and phenanthrene metabolites 2-hydroxyphenanthrene , 3-hydroxyphenanthrene , 4-hydroxyphenanthrene , and the sum of 1- and 9-hydroxyphenanthrene ., 13C-2-OHNa, 13C-2-OHFl, 13C-3-OHPH, 13C-4-OHPH, d8-9-OHPH) was added to the mixture to correct for the loss of analyte during sample preparation. Internal standards were provided by Campro Scientific GmbH, Veenendaal, the Netherlands, except for 13C-1-OHPy, which was provided by Sigma-Aldrich, Overijse, Belgium. After incubation, 240 µL acetonitrile was added to the mixture to keep the OH-PAHs in solution. An aliquot was transferred to an LC-vial and analyzed using ultra-high pressure liquid chromatography (UPLC) coupled to a tandem mass spectrometer  equipped with an Acquity BEH C18 column . Mass spectrometry was performed in negative ESI MRM mode, and two mass transitions were registered per analyte. All analytical lab operations were performed according to ISO 17025 from sample registration to reporting. The analytical method used was fully validated. Shewart charts were used to control quality over the total duration of the project. Warning limits were used as a trigger for reanalysis of the entire batch of the day. Practical internal quality control consisted of different measures. Analysis of blanks ensured absence of false positives. Random replicate sample analysis was used to control precision. In general, the relative standard deviation  ranged between 0 and 15%, where the higher values were recorded for samples with concentrations close to the LOQ. Repeated random analysis of spiked samples and of a control sample with standard reference material SRM 3672 [n = 23) was hereby recorded. External quality control was assured through successful participation in the Human Biomonitoring for Europe External Quality Assurance Scheme (HBM4EU ICI/EQUAS) for PAH metabolites [The analysis was performed at the Flemish Institute for Technological Research , using liquid chromatography coupled to mass spectrometry (UPLC–MS/MS). First, 500 μL of urine was hydrolyzed overnight at 37 °C with β-glucuronidase/arylsulfatase in the presence of a sodium acetate buffer (pH = 5) to obtain unconjugated OH-PAHs. A constant amount of internal standards for each analyte  kit , according to manufacturer’s instructions. The determination range was 0.5–200 ng/mL. The anti-8-oxodG mouse monoclonal antibody (clone N45.1), with an established specificity, was used as a primary antibody [For the determination of 8-oxodG, 1 mL urine was aliquoted, subsequently frozen, and stored at −80 °C until analysis at VITO Health. After thawing, samples were centrifuged at 2000× antibody . The valantibody .During fieldwork, a strand of hair of at least three centimeters was cut close to the scalp from the posterior vertex of the adolescents’ head. Hair samples were stored in paper envelopes at room temperature until analysis, within 18 months after the collection of the first samples. When protected from ultraviolet light, cortisol concentrations in hair samples remain stable at room temperature for several years . CortisoA blood sample of 6 mL was collected in an EDTA tube and gently mixed; 1 mL was then aliquoted in a Sarstedt tube. Samples were stored at 4 °C until analysis within 48 h after collection at A.M.L. Leucocyte count and leucocyte subtype distribution (percentage) were assessed using a Sysmex XE-2100 instrument for hematology analysis , a widely used automated hematology system that combines flow cytometry with fluorescence detection, using a diode laser bench . The neuStatistical analysis was performed using SPSS Statistics  and R version 3.5.0 . OH-PAHs concentrations, HCC, and 8-oxodG concentrations below the LOQ were imputed with a random value (between 0 and the LOQ), drawn from the estimation of the lognormal distribution of all values by fitting a truncated lognormal distribution using only values above the LOQ . Concentt-test for continuous variables and a chi-square test for categorical variables. Pearson’s correlations were calculated between all measured biomarkers. Biomarkers were related to population characteristics in univariate analysis.In addition, we calculated a sum parameter of all measured OH-PAHs (ΣOH-PAHs), which may serve as a proxy for total exposure to the measured OH-PAHs . First, Multiple linear regression models were constructed with each individual OH-PAH as an independent variable and each effect biomarker as a dependent variable. We specified three models with different levels of adjustment for a set of a priori defined potential confounders, based on literature ,55,56,57p ≤ 0.05 (two-sided). To quantify associations, the estimated factor change in the outcome variable (β) is presented with a 95% confidence interval (95% CI) for a doubling in OH-PAH concentration. As previously recommended [p ≤ 0.20 (two-sided).All assumptions for linear regression analysis were checked. The statistically significant level for associations was set at ommended , we inveTo check the robustness of our findings, we additionally adjusted our main models for neighborhood socio-economic status, using the area deprivation index (ADI). Studies suggest that neighborhood SES may influence health outcomes independent of personal SES . FurtherAs a secondary analysis, the effect of HCC on the relationship of OH-PAHs with leucocyte counts, NLR, and 8-oxodG was assessed for significant associations by adding HCC into each linear regression model and identifying changes in the relationship of OH-PAHs with leucocyte counts, NLR, and 8-oxodG. This is because the effect of OH-PAHs on inflammation and oxidative stress might be directly or indirectly through endocrine stress. Mediation models were constructed, using the Process-macro of Hayes  with HCCp = 0.002, 3.48  µg/L and 4.60  µg/L, respectively) and oftotal ΣOH-PAHs  µmol/L and 0.036  µmol/L, respectively). Geometric mean concentrations of other measured OH-PAHs did not significantly differ by sex . As presented in r > 0.50); the 2-ringed 2-OHNa correlated less strongly with 3- and 4-ringed OH-PAHs (Pearson’s r < 0.30). We found a strong correlation between ΣOH-PAHs and 2-OHNa (Pearson’s r = 0.99), the main contributor to the sum. p = 0.002, 6991  cells/µL versus 6471  cells/µL and p = 0.002, 3870  cells/µL versus 3186  cells/µL, respectively). NLR was also significantly higher in girls  versus 1.39 ). Geometric mean HCC was 2.85  pg/mg hair for boys and 3.26  pg/mg hair for girls, with no statistical difference by sex (p = 0.083). Further, geometric mean urinary 8-oxodG concentration did not significantly differ between boys and girls  µg/L and 17.00  µg/L, respectively, p = 0.790). As shown in r = 0.12) and NLR (Pearson’s r = 0.15). The urinary 8-oxodG concentration was not significantly correlated with HCC, NLR, or leucocyte counts. Univariate analyses of OH-PAHs and effect biomarker levels in relation to population characteristic and effect biomarkers are presented in Leucocyte and neutrophil counts were significantly higher in girls compared to boys  was estimated for a doubling in urinary 1-OHPy concentration. We did not observe significant associations between other measured urinary OH-PAHs or ΣOH-PAH and HCC.The urinary 1-OHPy concentration was significantly associated with HCC. As illustrated in p = 0.037) was estimated. The urinary concentration of 2-OHPH was significantly associated with total leucocyte count, neutrophil count, and NLR. A doubling in 2-OHPH concentration was associated with an increase in total leucocyte count with a factor of 1.03 , p = 0.048), an increase in neutrophil count with a factor of 1.06 , and an increase in NLR with a factor of 1.08 . A doubling in 3-OHPH was associated with a decrease in lymphocyte counts with a factor of 0.97 ; the association of 3-OHPH with NLR was borderline significant , p = 0.066). Urinary 1,9-OHPH concentration was inversely associated with lymphocyte count , p = 0.040).As shown in p = 0.007); a doubling in 2-OHPH and 3-OHPH concentration was associated with an increase in 8-oxodG with a factor of 1.08  and a factor of 1.06 , respectively. We did not observe significant associations of the ΣOH-PAHs with leukocyte counts or 8-oxodG.As illustrated in p-value of the interaction term) is presented in p-interaction = 0.067), 2,3-OHFl (p-interaction = 0.154), and 2-OHPH (p-interaction = 0.079). The interaction models, however, revealed no significant associations of the aforementioned OH-PAHs with HCC in boys nor girls. Furthermore, we observed significant heterogeneity of results by sex for the association of 2,3-OHFl with lymphocyte count and NLR . In girls, a doubling in urinary 2,3-OHFl concentration was associated with a significant decrease in lymphocyte count with a factor of 0.94 ; no significant change in lymphocyte count was observed in boys . The analysis also revealed that the significant association of 2-3-OHFl with NLR was driven by girls. A doubling in 2,3-OHFl concentration was associated with a significant increase in girls’ NLR with a factor of 1.10 . In boys, no significant changes in NLR were found in relation to urinary 2,3-OHFl concentration . Associations of OH-PAHs with 8-oxodG did not significantly differ by sex.Heterogeneity of results by sex  for a doubling in HCC; the association did not significantly differ by sex (p-interaction = 0.443). We observed no significant associations of HCC with 8-oxodG or leucocyte counts. Additional adjustment of our leucocyte, NLR and 8-oxodG models with HCC was of little influence on the strength of the observed associations. In particular, 2,3-OHFl and HCC were independently associated with NLR , p = 0.050 for 2,3-OHFl and β = 1.04 , p = 0.030 for HCC). Furthermore, we observed significant associations of both 2-OHPH and HCC with NLR , p = 0.011 and β = 1.05 , p = 0.027 respectively). The criteria for mediation analysis were not met for any of the observed associations.In a secondary analysis, we explored whether HCC was associated with leucocyte counts, NLR, and 8-oxodG and whether HCC was of influence on associations of OH-PAHs with these outcomes . We obseOur results indicate that exposure to PAHs may be associated with oxidative stress, inflammation, and chronic endocrine stress in a general population of adolescents. We evaluated all associations with different levels of adjustment for potential confounders. All results were robust to control for demographic , anthropometric (BMI), lifestyle (smoking and exposure to ETS) and meteorological factors (season and temperature). All potential confounders have previously been suggested to relate to exposure to PAHs ,38,55,64In line with the literature, we observed significant associations of OH-PAHs with the oxidative stress biomarker 8-oxodG. These findings may be relevant to public health, as oxidative damage to nucleic acids is a well-known initiator in the pathogenesis of cardiovascular diseases, neurodegenerative disorders, and cancer . Previoun = 660, 12–19 years), a significant association of 2-OHPH was observed with serum C-reactive protein levels in females only, whereas an increase in 9-hydroxyfluorene was related to an increase in CRP in males only [n = 2488), significant associations of the sum of urinary PAH metabolites  with leukocyte count and CRP were more evident among men than women [In addition, we observed significant associations of the phenanthrene metabolite 2-OHPH with leukocyte count, neutrophil count, and NLR, which are immune biomarkers of inflammation. The sum of fluorene metabolites  was also associated with NLR, with stronger associations observed in girls compared to boys. Differences in associations by sex between fluorene metabolites and inflammatory biomarkers have recently also been observed in the American National Health and Nutrition Examination Survey  . In thisles only . In adulan women . MoreoveThis study is the first to observe a significant association between urinary 1-OHPy and HCC as a chronic endocrine stress biomarker. Research on GC homeostasis in relation to exposure to EDCs has gained more attention recently ; howeverMaintenance of GC homeostasis is essential for the appropriate functioning of many cell types, including immune cells . In our Our study has several strengths. Urinary concentrations of PAH metabolites (OH-PAHs) reflect the integrated internal exposure through different routes of PAHs exposure, i.e., inhalation, ingestion, and dermal uptake . In FLEHIn this study, we did not observe significant associations between the sum of measured OH-PAHs and effect biomarkers. However, we found metabolites of different PAHs to be differentially associated with the studied biological mechanisms and observed differences in the strength of associations among metabolites of phenanthrene. Measuring different OH-PAHS may also provide more insight into PAH exposure. The OH-PAHs in this study all correlated to some degree, with correlations between three- and four-ringed fluorene, phenanthrene, and pyrene metabolites being stronger than correlations between these metabolites and the two-ringed naphthalene metabolite. Generally, the main source of exposure to naphthalene is through inhalation (mostly ambient pollution), and the sources of exposure to larger PAHs are more diverse  . AlthougSome limitations of the study also need to be addressed. PAHs that may co-vary with those assessed here but were not measured could have biologically relevant effects on our outcomes . In thisThis study indicates that higher urinary OH-PAHs concentrations are simultaneously but differently associated with increased levels of chronic endocrine stress, oxidative stress, and inflammation in a general population of Flemish adolescents. These findings may contribute to a better understanding of the pathways through which PAHs induce adverse health effects in adolescents.


Although bone marrow stimulation (BMS) as a treatment for osteochondral lesions of the talus (OCLT) shows high rates of sport resumption at short-term follow-up, it is unclear whether the sports activity is still possible at longer follow-up. The purpose of this study was, therefore, to evaluate sports activity after arthroscopic BMS at long-term follow-up.Sixty patients included in a previously published randomized-controlled trial were analyzed in the present study. All patients had undergone arthroscopic debridement and BMS for OCLT. Return to sports, level, and type were assessed in the first year post-operative and at final follow-up. Secondary outcome measures were assessed by standardized questionnaires with use of numeric rating scales for pain and satisfaction and the Foot and Ankle Outcome Score (FAOS).The mean follow-up was 6.4 years (SD ± 1.1 years). The mean level of activity measured with the AAS was 6.2 pre-injury and 3.4 post-injury. It increased to 5.2 at 1 year after surgery and was 5.8 at final follow-up. At final follow-up, 54 patients (90%) participated in 16 different sports. Thirty-three patients (53%) indicated they returned to play sport at their pre-injury level. Twenty patients (33%) were not able to obtain their pre-injury level of sport because of ankle problems and eight other patients (13%) because of other reasons. Mean NRS for pain during rest was 2.7 pre-operative, 1.1 at 1 year, and 1.0 at final follow-up. Mean NRS during activity changed from 7.9 to 3.7 to 4.4, respectively. The FAOS scores improved at 1 year follow-up, but all subscores significantly decreased at final follow-up.At long-term follow-up (mean 6.4 years) after BMS for OCLT, 90% of patients still participate in sports activities, of whom 53% at pre-injury level. The AAS of the patients participating in sports remains similar pre-injury and post-operatively at final follow-up. A decrease over time in clinical outcomes was, however, seen when the follow-up scores at 1 year post-operatively were compared with the final follow-up.Level II.The online version of this article (10.1007/s00167-020-06250-8) contains supplementary material, which is available to authorized users. Osteochondral lesions of the talus (OCLT) are lesions to the subchondral bone and the overlying cartilage layer. These injuries have a high association with inversion injuries and ankle fractures , 21, 29.As OCLTs are highly frequently seen in the athletic population, it is necessary to put specific emphasis on outcomes of treatment in athletes . A recenIn most studies, it is often unclear whether authors reported on return to pre-injury level or return to any associated level of sports. Furthermore, there is a significant deficiency reported in rehabilitation protocols , 27. EspThe study has been approved by the local medical ethics committee at the University of Amsterdam and was performed in accordance with the current medical ethical standards . Written informed consent for participation was obtained from every patient. We included the patient cohort of Reilingh et al. and adhered to the inclusion and exclusion criteria of this previously published study Fig. .Fig. 1StPatients included in a previously trial, the Pulsed Electro Magnetic Fields (PEMF) trial, were analyzed in the present study . In thatPatients followed a protocol-based rehabilitation program post-operatively and were guided by a physiotherapist. The patient was allowed to progress to full weight-bearing in 6 weeks. During this 6-week period, active nonweight-bearing and partial weight-bearing sagittal range of motion exercises were encouraged. After this period, resumption of sports was permitted as tolerated .© portal. To reach all patients from the previous PEMF trial, consisting of a cohort of 68 patients, a personalized email was sent out to the potential subjects of the study [Clinical outcome assessment of the patients was performed by reaching out to the patients, requesting them to fill out an online questionnaire via the CASTORhe study . In casen = 53), 3 working activities, and 4 general activities [Through the online post-operative questionnaire, return to sports was assessed, including type and level of sports. Concerning sports activities, the Ankle Activity Scale (AAS) was used . The AAStivities . Scoringtivities . The scotivities . Furthertivities , the Footivities , 24, wortivities .The sport outcomes, the AAS, the NRS scales (at rest and when running), the FAOS, and return to work had also been assessed in the previous study by Reilingh et al.  both preAll analyses were done using Statistical Packages for Social Sciences . To minimize the risk of attrition bias, we included all patients with a completed electronic questionnaire at final follow-up in the statistical analyses. This includes the patients who underwent a reoperation. No additional sample size calculation was performed as this study was a follow-up study of a previously published study. Detailed information on the sample size calculation is described in this previous study . Descripp < 0.05 were considered to be statistically significant.Descriptive statistics of continuous variables were calculated as means ± standard deviations or median and ranges in case of a skewed distribution. Categorical variables were presented as frequencies with percentages. Differences of the continuous outcome variables were assessed between baseline and final follow-up and between 1 year follow-up and final follow-up. In case of normally distributed data, a paired t-test was used; in case of skewed data, a Wilcoxon signed-rank test was used. The comparisons with Four patients of the 64 (6%) were lost to follow up and could not be reached. The other 60 patients were available for follow-up. No patients declined to participate. The mean post-operative follow-up was at 6.4 years after surgery (SD ± 1.1 years). Mean age of the patients at follow-up was 39 years (SD ± 8.5 years).A sub-analysis of the original groups (the PEMF group and the placebo group) showed no significant difference at any of the outcome measures . Data could thus be interpreted as one group.Of all included patients, all participated in sports before injury of which 6 on a professional level, 29 on a competitive level, and 25 on a recreational level. The median pre-injury ankle activity score of the 60 included patients was 6.2 (range 4–8). After onset of complaints pre-operatively, only 27 patients were still participating in sports of which 2 on a professional level, 6 competitive, and 19 recreational. The other 33 were not able to participate in sports anymore. The AAS decreased to 3.4 post-injury. After surgery, the median time to return to sports of the 60 included patients was 18 weeks. At 1 year after surgery, 85% of patients returned to playing sport and the AAS increased to 5.2. At final follow-up, 90% of patients participated in sports. These 54 patients participated in 16 different sports, some participating in more than one sport. Most played sport was soccer (16 patients) followed by running (14 patients), fitness (12 patients), cycling/mountainbiking (9 patients), and tennis (7 patients). Of all patients playing sports at follow-up, 35 patients played at a recreational level and 18 patients played at a competitive level. One patient played table tennis on a professional level for years after surgery. At final follow-up, the median current level of activity of all the patients measured with the AAS was 5.8 (range 1–9)  indicated that they returned to play sport at their pre-injury level. Twenty patients (33%) were not able to obtain their pre-injury level of sport because of ankle problems despite the operation. Another eight patients (13%) indicated that they were not able to play sport at their pre-injury level because of different reasons than their ankle.Three patients underwent 4 reoperations in total. One patient received an ankle arthrodesis at 6 years after the original surgery because of progressive osteoarthritis and experienced pain. One patient received a reoperation due to posterior impingement 15 months after the original surgery. This and another patient also underwent an adjacent procedure in another hospital.Twenty-six patients experienced one or more additional injuries, e.g., back pain or spine injuries (5 patients), anterior cruciate ligament tears (4 patients), other knee problems (5 patients), and achilles tendon problems (3 patients).Fifty-eight patients resumed to their pre-injury work. One patient indicated that he worked less due to persisting ankle problems. Seven worked less due to other reasons. Four patients reported to be working more because of a decrease in ankle problems.p < 0.01) and the NRS pain during activity from 7.9 to 4.3 (p < 0.01). The FAOS other symptoms decreased from 64 to 52 (p < 0.01), the FAOS pain increased from 61 to 67 (p = 0.01), the FAOS ADL from 66 to 79 (p < 0.01), the FAOS sport from 41 to 48 (n.s.), and the FAOS QOL from 29 to 44 (p < 0.01).The clinical outcomes (NRS and FAOS scores) are presented in Figs. p < 0.01), the FAOS pain from 82 to 67 (p < 0.01), the FAOS ADL from 86 to 78 (p = 0.02), the FAOS sport from 67 to 48 (p < 0.01), and the FAOS QOL from 54 to 44 (p < 0.01).Between 1 year and final follow-up, the NRS did not change significantly with the NRS during rest from 1.1 to 1.0 (n.s.) and the NRS activity from 3.7 to 4.3 (n.s.). All the FAOS scores, however, significantly decreased. The FAOS other symptoms decreased from 70.6 to 52 .The most important finding of the present study was that arthroscopic BMS in OCLT results in 90% of return to sports/physical activity with 53% being able to return to their pre-injury level of sport at a mean follow-up of 6.4 years. This percentage of sports participation is comparable with follow-up at 1 year post-operatively. The AAS of the patients participating in sports remains similar pre-injury and post-operatively at final follow-up. However, a decrease over time in clinical outcomes were seen when we compare the FAOS follow-up scores at 1 year post-operatively with the final follow-up.Return to sports is important in most young and active patients and of course of uttermost importance in professional athletes. The sustainability of the repaired cartilage is essential in this group to remain active at the highest level. High reported RTS rates at pre-injury level have been reported after BMS in the ankle of up to 94% in elite athletes after a mean follow-up of 3.6 years . SimilarWith multiple techniques being available, bone marrow stimulation still is worldwide the most performed intervention for both primary and secondary osteochondral lesions of the talus , 14. It The present study has to be interpreted in light of its strengths and weaknesses. Since four patients were lost to follow up from a group of 64 patients, the lost to follow-up percentage is calculated to be 6%. An acceptable lost to follow up, however, according to the Center for Evidence Based Medicine. Furthermore, since the lost to follow up presumable is missing at random after a mean follow-up period of more than 6 years, we think that it is an adequate follow-up and representative study group . AnotherThe results of this study can be used to inform patients, specifically active patients, about expectations around long-term sports outcome after bone marrow stimulation. It also shows that although there is a high return to sport participation, there is still room left for improvement, since outcome scores decreased over time. Possible further research could focus on sport outcome after treatment types that are more based around the preservation of hyaline cartilage such as transplantation or fixation techniques.Arthroscopic BMS in OCLT results in 90% of return to sports/physical activity with 53% being able to return to their pre-injury level of sport at a mean follow-up of 6.4 years. The percentage of sports participation is comparable with follow-up at 1 year post-operatively. The AAS of the patients participating in sports remains similar pre-injury and post-operatively at final follow-up. A decrease over time in clinical outcomes was seen at final follow-up.Supplementary file1 (DOCX 70 kb)Below is the link to the electronic supplementary material.


The use of Akt inhibitors or FOXO3a agonists, such as PD, may represent a promising approach for the treatment of androgen-independent and PTEN-deficient prostate cancer.Castration-resistant (androgen-independent) and PTEN-deficient prostate cancer is a challenge in clinical practice. Sorafenib has been recommended for the treatment of this type of cancer, but is associated with several adverse effects. Platycodin D (PD) is a triterpene saponin with demonstrated anti-cancer effects and a good safety profile. Previous studies have indicated that PC3 cells  are sensitive to PD, suggesting that it may also be a useful treatment for castration-resistance prostate cancer. We herein investigated the effects of combining PD with sorafenib to treat PTEN-deficient prostate cancer cells. Our data show that PD promotes sorafenib-induced apoptosis and cell cycle arrest in PC3 cells. Of interest, PD only promoted the anti-cancer effects of sorafenib in Akt-positive and PTEN-negative prostate cancer cells. Mechanistic studies revealed that PD promoted p-Akt ubiquitination by increasing the p-Akt level. PD also increased the protein and mRNA expression of FOXO3a, the downstream target of Akt. Meanwhile, PD promoted the activity of FOXO3a and increased the protein expression of Fasl, Bim and TRAIL. Interestingly, when FOXO3a expression was inhibited, the antitumor effects of both PD and sorafenib were individually inhibited, and the more potent effects of the combination treatment were inhibited. Thus, the combination of PD and sorafenib may exert potent anti-cancer effects specifically  According to 2019 data, the incidence and death rates of prostate cancer are first and second in men in the USA , 4. ResePTEN, a tumor suppressor gene, is related to cancer progression and malignancy, and its loss is associated with a poor prognosis –9. Approin vivo observations.Sorafenib is a molecular targeted drug approved for the treatment of several human cancers. Steinbild et al. showed that sorafenib is beneficial for patients with progressive hormone-refractory prostate cancer . Howevervia its transcriptional regulation of key genes involved in cell cycle arrest, apoptosis, DNA damage and drug resistance  of action for PD, alone and in combination sorafenib, in PTEN-deficient and androgen-independent prostate cancer.PD was purchased from Must Bio-Technology Co., Ltd.  and sorafenib was purchased from Selleckchem . Fetal bovine serum (FBS) was purchased from Bioind . The anti-FOXO3a, anti-FasL, anti-Bim, anti-TRAIL, anti-Akt and anti-Ubiquitin antibodies were purchased from Cell Signaling Technology, Inc. . The anti-p-Akt, anti-ERK and anti-p-ERK antibodies were obtained from Abcam . The anti-Caspase 3, anti-Cleaved Caspase 3, anti-Caspase 3 and anti-PARP antibodies were obtained from Beyotime Biotechnology, Inc. . The anti-CDK4, anti-CDK6 and anti-cyclin D antibodies were obtained from Boster Biological Technology, Inc. . The Z-VAD-FMK, MG132, pictilisib and MK2206 were purchased from Selleckchem . Lipofectamine™ 2000 was purchased from Invitrogen . The shRNA-FOXO3a plasmid, cDNA-Akt plasmid and cDNA-FOXO3a were purchased Shanghai GeneChem Co., Ltd .The PC3 (PTEN-deficient) and DU145 (PTEN wild type) cell lines were purchased from the Chinese Academy of Sciences . The cell culture procedures have been described in our previous papers . In brie5 or 8*104 cells. Cells were transduced with the shRNA, cDNA, empty lentivirus or empty plasmid at a multiplicity of infection in accordance with the manufacturer’s instructions. Puromycin or hygromycin B were used to select stably-transfected cells during viral transduction. Western blotting was carried out to verify the knockdown efficiency.Lentiviruses encoding shRNA against PTEN and FOXO3a, cDNA for PTEN and Akt, and plasmids for Akt and FOXO3a, were constructed by Shanghai GeneChem Co., Ltd. . Empty lentivirus and plasmid constructs were used as the negative controls. Cells were cultured in T25 flasks or 6-well plates at 6*10Cell survival , FITC/PIThe CFDA SE assay was used to detect the effects of PD and Sorafenib on the proliferation of PC3 cells over time. PC3 cell suspensions were labeled with CFDA-SE, then were plated into 6-well plates overnight. The cells were then collected after being treated with the compound(s) for 7 days. The samples were analyzed for staining using a FACSCaliber flow cytometer.Cells were implanted in 6-well plates (1000 cells/well). After treatment with different concentrations of PD and Sorafenib for 10 days, the cells were fixed with paraformaldehyde and stained with crystal violet. The samples were then photographed and counted using Photoshop.The methods used for RNA extraction, reverse transcription-PCR and real-time quantitative PCR have been described in previous articles . In brieThe cells were treated with various concentrations of PD for 10 min following pretreatment with 10 µM MG132 for 6 h. Whole cell lysates were obtained by cell lysis in ice-cold RIPA buffer. After lysis, samples were shaken on a horizontal shaker for 30 min with 50% protein A/G agarose at a ratio of 100 µl per 1 ml of sample solution. After shaking, the samples were centrifuged at 14,000g at 4°C for 15 min, then the supernatant was transferred to new tubes and the protein A/G-agarose beads were discarded. At that time, 3 μl of anti-Akt antibody was added to approximately 500 μl total volume, and samples were kept at 4°C overnight. The next morning, 50% protein A/G agarose was added to the sample solution, and samples were kept at 4°Cfor 1 h. The samples were then centrifuged at 3000 r/min for 2 min. The pellet was retained and washed with pre-iced RIPA buffer 3 times for 30 min each time. The resulting pellets were kept for the subsequent Western blot analyses.Whole cell lysates were obtained by the lysis of prostate cancer cells or tumor tissue as described previously . The extvia the transfection of a shRNA-FOXO3a lentivirus. The resulting PC3 cells (shRNA-FOXO3a cells and MOCK (control) cells) were injected subcutaneously into the left inguinal area of nude mice . One week after tumor cell inoculation, mice bearing palpable tumors were randomly divided into control and treatment groups (6 mice/group). The PD was dissolved in the vehicle, PEG400:Saline : Ethanol , and administered (via i.p. injection) at doses of 2.5 mg/kg body weight, 5 consecutive days a week for 30 days. The control group received injections of the vehicle. The mice were sacrificed by cervical dislocation on day 30, and the tumor tissues were removed and weighed. All animals were monitored for activity, general condition, body weight, and tumor growth. The tumor size was measured every third day using calipers, and the tumor volume (cm3) was calculated by the following formula: (a *b2)/2, where ‘a’ and ‘b’ represent the longer and shorter dimensions.Mouse xenograft models have been described in previous articles . In shorp< 0.05.The data for the different treatment groups are presented as the means ± standard errors. A one-way ANOVA was used to determine the significance of the effects of the treatments on the levels of apoptosis or cell death. The results were considered to be statistically significant for values of As shown in We also analyzed the changes in PC3 cell morphology after cells were treated with 10 μM PD alone, 10 μM sorafenib alone or PD plus sorafenib for 24 h. SEM and TEM showed that the cell membranes and nuclei were more damaged in cells treated with the combination of PD and sorafenib than in those treated with either agent alone , antibiotic (clofoctol) or a natural product (Nobiletin) promoted the anti-cancer effects of sorafenib in castration-resistant prostate cancer –49. Thesvia suppression of Akt-associated pathways   . Under tTTCA-3’) . Therefovia a mechanism dependent on FOXO3a, and that it promoted FOXO3a function through pathways involving molecules other than Akt. This may be one of the reasons why sorafenib promoted the effects of an AR inhibitor in AR-dependent prostate cancer. However, FOXO3a has been shown to react differently to sorafenib in different cell types. For example, sorafenib up-regulated the protein expression of FOXO3a in B- and T-lymphoblastic cells (Research by Yamamoto and colleagues showed that a single dose of sorafenib (30-60 mg/kg) decreased the activation of the PI3K/AKT/mTOR signaling axis in a mouse model of PTEN-deficient prostate cancer . We showic cells  and downic cells . Thus, tIn summary, our present findings provide evidence that PD increases the ubiquitination of Akt. PD also promotes the protein and mRNA expression of FOXO3a. We demonstrated that FOXO3a-mediated effects appear to play an important role in PD-induced prostate cancer cell death. Moreover, the increase in FOXO3a function caused by PD promoted the anti-tumor effects of sorafenib. Therefore, PD  can be used for the treatment of PTEN-deficient and androgen-independent prostate cancer. Further studies will be needed to elucidate the detailed mechanisms underlying these findings. However, the current results provide support for further studies of PD as a candidate natural product-based preventive, auxiliary and/or therapeutic agent for prostate cancer.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.HX and ZL designed research. ZL, WS, YWZ, and CPW performed research and wrote the paper. MZ, HW, and YZ analyzed data. MZ contributed the statistical analysis. NL contributed the reagents. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (Nos. 81603347), the Natural Science Foundation of Chongqing (cstc2020jcyj-msxmX0499), and a Basic Science and Frontier Technology Research Project of Chongqing (CSTC2017jcyjAX0305).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both the chamber atmospheres and the device passivation layers proved important for the post-annealing effects on a-IGZO TFTs. For the heat treatments in O2 or air, the larger threshold voltage (VTH) and off current (IOFF), smaller field-effect mobility (μFE), and slightly better PBS stability of a-IGZO TFTs were obtained. The X-ray photoemission spectroscopy (XPS) and secondary ion mass spectroscopy (SIMS) measurement results indicated that the oxygen atoms from the ambience led to less oxygen vacancies (VO) and more oxygen-related defects in a-IGZO after the heat treatments in O2 or air. For the annealing processes in vacuum or N2, the electrical performance of the a-IGZO TFTs showed nearly no change, but their PBS stability evidently improved. After 4500 seconds’ stressing at 40 V, the VTH shift decreased to nearly 1 V. In this situation, the SiOx passivation layers were assumed to effectively prevent the oxygen diffusion, keep the VO concentration unchanged and refuse the oxygen-related defects into the a-IGZO films.We investigated the electrical performance and positive bias stress (PBS) stability of the amorphous InGaZnO thin-film transistors (a-IGZO TFTs) with SiO O), one of the most important defects in a-IGZO films [O was sensitive to annealing atmospheres, as some researchers obtained higher performances and stabler properties of a-IGZO TFTs by choosing appropriate annealing atmospheres  [Amorphous InGaZnO thin-film transistors (a-IGZO TFTs) have been regarded as among the most promising active-matrix devices for next-generation flat panel displays (FPDs) due to their high mobility, good uniformity and low fabrication temperature ,2,3,4,5.ZO films ,13. Assud so on) ,15,16,17x passivation layers under various post-annealing atmospheres  were comparatively investigated, whose physical essence was deeply analyzed with the help of X-ray photoemission spectroscopy (XPS) and secondary ion mass spectroscopy (SIMS). We found that both the chamber atmospheres and the device passivation layers played important roles in the post-annealing treatments for a-IGZO TFTs. A qualitative model was also built to explain the experimental results.In this study, the electrical performance and positive bias stress (PBS) stability of the a-IGZO TFTs with SiOx and 50 nm-thick SiOx films were prepared as gate insulators (GIs) by plasma-enhanced chemical vapor deposition (PECVD). The 60 nm-thick channel layers were deposited by RF sputtering at room temperature (RT) using an IGZO target, where the atoms ratio of In:Ga:Zn was 1:1:1. After patterning the channel layers, a pre-annealing treatment (300 °C/1 h/air) was employed in a furnace, which could increase the film density/uniformity and reduce the defects in the a-IGZO films. The 30 nm-thick molybdenum and 150 nm-thick aluminum films were then prepared and patterned as source/drain (S/D) electrodes before the 200 nm-thick SiOx films were deposited as passivation layers by PECVD. Finally, the samples were annealed at 300 °C for 1 h in different atmospheres  using a rapid thermal processing (RTP) oven, where the gas flows of O2 (or N2) were set to 1 L/min and the vacuum pressure was kept at 6.5 × 10−3 Pa. It is worth noting that all the films were patterned by standard lithography and etching processes. The typical channel width/length of the a-IGZO TFTs was 12/6 μm.GS was scanned from −20 V to 40 V at a step of 0.5 V and VDS was set to 10 V. During the PBS tests, the transfer curves were instantly measured following each 1500 s’ stressing (VGS = +40 V). All the above tests were performed in a dark chamber at RT. An XPS analyzer (AXIS Ultra DLD) was used to characterize the chemical bonding states of the a-IGZO films (without or with passivation layers) annealed under different atmospheres. Here, an X-ray source with the aluminum anode was used to bombard the film surface and obtain the energy spectrum of different elements. The binding energy of all elements were calibrated using the C1s peak at 284.8 eV. A SIMS measurement system (TOF SIMS 5 produced by ION-TOF GmbH) with a Cs+ primary ion source was applied to measure the depth profiles of different elements.The electrical performance and PBS stability of the a-IGZO TFTs were measured by a Keithley 2636 analyzer. The transfer curves were obtained when VTH), field-effect mobility (μFE), sub-threshold swing (SS) and on–off current ratio (ION/IOFF) were extracted from the transfer curves according to Ref. [2, N2 and vacuum) were 3.07, 2.08, 1.37, 3.78 and 4.03 cm2/(V·s), respectively. One may notice that the annealing in O2 or air obviously degraded the device mobility, whereas the annealing in N2 or vacuum slightly improved μFE. In addition, the corresponding VTH values were 3.6, 5.1, 8.0, 4.7 and 5.4 V, the SS values were 2.8, 2.4, 2.7, 2.6 and 2.4 V/dec, and the on–off current ratios were 3.8 × 106, 2.4 × 106, 3.9 × 105, 8.2 × 106 and 1.3 × 107, respectively  except that in O2, as shown in  to Ref. . Here, 6vely see b,c. Compx passivation under various annealing atmospheres . TH  in this study. It is worth noting that each ΔVTH value was averaged over 3–6 devices. After 4500 s’ stressing, ΔVTH of the unannealed device became 3.67 V. All the post-annealed devices exhibited better bias stress stability than the unannealed sample. It is interesting that the ΔVTH value distinctively depended on the post-annealing atmosphere. As shown in TH decreased to 1.88 V, 2.50 V, 1.13 V and 1.38 V for the devices annealed in air, O2, N2 and vacuum, respectively. It is worth noting that the post-annealing treatments in N2 and vacuum showed much larger improvements than those in O2 and air.The bias stress effect plays an important role in the actual applications of TFT devices ,19. AccoI), 530.9 eV (OII) and 532.0 eV (OIII), respectively. The OI peak represents the oxygen bonds with metal, the OII peak is bound up with VO, and the OIII peak is related to the hydrated oxides defects [II and OIII over the total area of O1s peak . The area ratio OII/OTotal of the a-IGZO films annealed under various atmospheres  were 28.56%, 28.19%, 21.78%, 30.20% and 30.02%, respectively. One may observe that the post-annealing in O2 largely decreased the area ratio OII/OTotal, whereas the heat treatments in air/N2/vacuum showed little effect in this regard. On the other side, the area ratio OIII/OTotal of the a-IGZO films under different atmospheres  were 13.08%, 16.91%, 17.19%, 17.53% and 17.37%, respectively. It is obvious that all the post-annealing treatments increased the area ratio OIII/OTotal.In order to determine the related physical mechanisms, we used the XPS technique to analyze the a-IGZO films (without or with passivation layers) annealed under various atmospheres. Firstly, we deposited 60 nm-thick a-IGZO films on glass substrates, annealed them under different atmospheres, and applied them to XPS measurements. Although these unpassivated films were different from the real situation in the TFT devices see , they co defects ,21. Figu2_annealing among five treatments evidently changed VO in a-IGZO films, which are in need of much further study. Most importantly, could passivation layers prevent this effect? Attempting to address this question, we used XPS depth profiling technique to analyze the chemical bonding states in the a-IGZO films passivated by 50 nm-thick SiOx [x, whereas point B was in the middle of a-IGZO films. The characterization results of the unannealed samples and the O2_annealed samples are shown in II/OTotal decreased from 31.0% to 25.8% by the O2_annealing treatment, which was quite similar to the case of the unpassivated a-IGZO films  and O2_annealed (34.8%) samples. These results indicated that the oxygen atoms might also diffuse into the a-IGZO back surfaces and hence combine with VO even if the SiOx passivation layer was applied during the O2_annealing treatments. Although the thickness of SiOx used here (50 nm) was thinner than that of the passivation layer (200 nm) in the a-IGZO TFTs, we assume that this conclusion was also applied to the real devices.As shown in ick SiOx . As showilms see b. At poi2_annealed sample, both of which consisted of 60 nm-thick a-IGZO and 160 nm-thick SiOx films. Here, the SIMS depth profiling technique was used to analyze the distribution of different elements in the unannealed and O2-annealed samples [x and a-IGZO. The O2_annealing brought nearly the same SIMS picture as that of the unannealed sample except the data about oxygen ions. As shown in x films by the O2_annealing, implying that the entering oxygen atoms from the ambience during the annealing treatment in O2 were too few to be detected by SIMS. Therefore, we reasonably assume that the oxygen atoms entering the a-IGZO also could not be detected by SIMS. However, as shown in 2_annealed sample. We assume that the microstructure of the a-IGZO evidently changed during the annealing treatment in O2, which might be due to the entering oxygen atoms from the ambience although their concentration was quite small. This change also implied that the O2_annealing might bring some oxygen-related-defects such as interstitial oxygen in the a-IGZO films [In order to further ascertain the role of the ambient oxygen atoms during the thermal annealing treatments, we comparatively measured the SIMS profiles of the unannealed sample and O samples . Figure ZO films ,25.2 (or air) atmospheres. In this situation, the oxygen atoms were full of the RTP chamber, so the oxygen diffusion from the chamber into the a-IGZO films should prevail that from the device channel layers into the ambience. It was reported that oxygen atoms could pass through dielectric films due to thermal diffusion at high temperatures [O concentration in a-IGZO films, which was confirmed by the XPS measurement results . In this situation, there were few oxygen atoms in the RTP chamber, so the oxygen diffusion from the a-IGZO film to the ambience was assumed to be dominant. If so, the VO concentration would have become larger. However, this rise in the VO concentration was not apparent due to the XPS measurement data. As shown in 2 (or vacuum) hardly changed the area ratio OII/OTotal. We ascribe this phenomenon to the barrier effect of the SiOx passivation layers. In contrast to x passivation layers effectively prevented the oxygen diffusion from the a-IGZO into the ambience during heat treatments in vacuum (or N2). Since the VO concentration changed little in this case, the electron concentration in the channel layers of the a-IGZO TFTs remained nearly unchanged, leading to VTH and μFE values similar to those of the unannealed device (see 2), we reasonably assume that there were no oxygen-related-defects included here, so the improvement effect over the stable properties of a-IGZO TFTs by heat treatments could be well kept [2) effectively improved the PBS stability of the a-IGZO TFTs with SiOx passivation layers, which should be preferred in mass productions.vice see b. As forell kept . As showO and the other one is relating the choice of passivation materials. Some researchers reported the VO drop led to better device performance [2_annealing treatments, i.e., they not only decreased the VO concentration but also brought some oxygen-related-defects [x was selected as passivation layers, which was found to play important roles in the post-annealing treatments under different atmospheres. We assumed that other passivation materials should bring similar tendencies but to different extents. This assumption might be supported by the other research results from our group, which indicated that the a-IGZO TFTs with SiOx and AlOx passivation layers exhibited almost the same PBS stability if the different passivation layer thicknesses were used [O, leading to better thermal stability [Finally, we briefly discussed two interesting questions relating this study. The first one is about the function of Vformance ,36, whic-defects ,25. In tere used . In facttability . Althougx passivation layers. For the heat treatments in O2 (or air), the oxygen atoms in the ambience diffused into the a-IGZO and combined with oxygen vacancies, leading to less VO and more oxygen-related defects; this resulted in larger VTH/IOFF, smaller μFE and the slightly better PBS stability of the a-IGZO TFTs. For the post-annealing process in vacuum (or N2), the oxygen diffusion was effectively prevented by the SiOx passivation layers, and hence the VO concentration hardly changed; the electrical performance of the a-IGZO TFTs showed nearly no change, but very importantly, their PBS stability evidently improved. This qualitative model was confirmed by the XPS and SIMS measurement results.The post-annealing atmosphere effectively influenced the electrical performance and bias stress stability of the a-IGZO TFTs with SiO


A proportion of patients with immunogloblin G (IgG) 4-related disease (IgG4-RD) have hypocomplementemia. We aimed to identify characteristics of such patients.We analyzed the demographic and clinical data and complement levels of 85 patients with IgG4-RD. We defined hypocomplementemia as serum C3 and/or C4 levels below the lower limit of normal at diagnosis. We also compared the characteristics of patients with and without IgG4-RD.Thirty-two (38%) patients had hypocomplementemia at diagnosis. Patients with hypocomplementemia had more lymph node (p < 0.01), lung (p < 0.01), and kidney (p = 0.02) involvement and a higher IgG4-RD responder index than those without (p = 0.05). Additionally, patients with hypocomplementemia had significantly higher IgG (p < 0.01), IgG4 (p < 0.01), and soluble interleukin 2-receptor (sIL-2R) (p < 0.01) levels and total IgG minus IgG4 (p < 0.01). C3 and C4 levels negatively correlated with IgG, IgG4, and sIL-2R levels, total IgG minus IgG4, and number of IgG4-RD responder index: a measure of the disease activity in IgG4-RD. Patients with hypocomplementemia at diagnosis had a significantly higher frequency of relapse (p = 0.024), as determined using the log-rank test. A multivariate logistic regression analysis showed the presence of hypocomplementemia was independently associated with relapse .Patients with IgG4-RD with hypocomplementemia have a more active clinical phenotype, suggesting contributions of the complement system in the pathophysiology of IgG4-RD. Involved tissues exhibit IgG4-positive lymphoplasmacytic infiltrates and fibrosis on histopathology . IgG is phocytes .Hypocomplementemia, defined as having C3 or C4 complement levels below the lower limits of normal, is one of the serological features of IgG4-RD. More than one-third of Japanese patients with IgG4-RD have hypocomplementemia . In a smA total of 107 patients diagnosed with IgG4-RD between December 2008 and September 2021 were recruited from the Department of Immunology and Rheumatology of Nagasaki University Hospital, the Department of Rheumatology of Fukushima Medical University Hospital, and affiliated hospitals. Twenty-two IgG4-RD patients whose complement levels were not measured at diagnosis were excluded. All patients fulfilled the 2020 revised comprehensive diagnostic criteria for IgG4-RD . Disease activity was determined based on the IgG4-RD responder index (IgG4-RD RI) . We obtath–75th centiles . Spearman’s correlation coefficient was used to evaluate correlations. The chi-square test was used to compare categorical variables represented as frequencies. The relapse-free survival analysis was assessed using Kaplan–Meier analysis, with the significance based on the log-rank test.Data were analyzed with SPSS Statistics software version 22.0 . Results were nonnormally distributed and compared with the Mann–Whitney U test and are presented throughout the manuscript with median and 25We compared the characteristics of patients with IgG4-RD with hypocomplementemia (n = 32) to those without (n = 53). Categorical variables are described as frequencies, whereas quantitative variables are described as medians and IQR. Associations between the variables were assessed using Fisher’s exact test for categorical variables and Wilcoxon’s rank-sum test for quantitative variables. Relationships between variables were analyzed using Spearman’s rank correlation. The prognostic factors for relapse were identified using a stepwise multiple logistic regression model. All tests were two-sided, and p-values of <0.05 were considered significant.The study was approved by the Institutional Review Board of Nagasaki University Hospital (17091109), Fukushima Medical University (29317), and affiliated hospitals. Informed consent for data use was obtained from some of the patients, and an opt-out procedure was used for the rest.The demographic and clinical characteristics of all 85 patients with and without hypocomplementemia at diagnosis are summarized in Thirty-two patients (38%) were classified in the hypocomplementemia group. In the hypocomplementemia group, 22 patients (69%) had C3 and C4 reduction, 3 (9%) had only C3 reduction, and 7 (22%) had only C4 reduction.The median age at diagnosis was 66 years; 62 patients were men (73%). There were no significant differences in age at diagnosis, sex, body mass index, and history of allergy between the two groups.The hypocomplementemia group had significantly higher IgG  , lung , and kidney  involvement. Patients with hypocomplementemia also had a significantly higher IgG4-RD responder index  Table 1.The correlations between C3 levels, C4 levels, and other biological measures are presented in Kaplan–Meier survival analysis on disease relapse is shown in Hypocomplementemia was previously reported to occur concomitantly with IgG4-RD , 11. Thivia the classical pathway  . C5a recIgG4-RD is generally treated using corticosteroid, but the treatment other than corticosteroid has not been established. Recent report has shown that C5a receptor inhibitor is effective in ANCA-associated vasculitis . HoweverBased on these considerations, the hypothesis of pathophysiology in IgG4-RD is shown in Our study had some limitations. First, it was a retrospective study of a relatively small number of patients, thereby the number of explanatory variables in multivariable analysis may be limited. In addition, the other complement factor including C5a could not be measured because our sample were stored at -20°C. which is unstable condition for complement factors.Second, the proportion of patients with pancreas and kidney involvement was small; the patients had been referred from the Department of Rheumatology, thereby potentially leading to selection bias regarding the involved organs. Third, evaluating the extent of IgG4-RD only by the number of affected organs and IgG4-RD RI may be insufficient to assess disease activity because of the differentiation of radiological modality in each patient. The measurement of other biomarkers such as plasmablasts  and CC-cIn conclusion, our study revealed that patients with IgG4-RD with hypocomplementemia had more serious disease course than those with normal complement levels. Furthermore, the strong treatments may be considered for IgG4-RD patients with hypocomplementemia.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of Nagasaki University Hospital. The patients/participants provided their written informed consent to participate in this studyYF made substantial contributions to the study concept and design with the assistance of SF. YF conducted the statistical analyses and drafted the manuscript with the assistance and supervision of KM and AK. YF, MU, ST, NI, YN, YH, TS, AO, TA, YU, AM, TO, and HW treated the patients and collected the primary data. AK critically revised the manuscript, supervised the entire study, and gave final approval to the article. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.